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Am J Physiol Endocrinol Metab 291: E1017-E1024, 2006. First published June 20, 2006; doi:10.1152/ajpendo.00140.2006
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Long-lived {alpha}MUPA transgenic mice exhibit pronounced circadian rhythms

Oren Froy,1 Nava Chapnik,1 and Ruth Miskin2

1Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, Rehovot; and 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Submitted 23 March 2006 ; accepted in final form 16 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Robust biological rhythms have been shown to affect life span. Biological clocks can be entrained by two feeding regimens, restricted feeding (RF) and caloric restriction (CR). RF restricts the time of food availability, whereas CR restricts the amount of calories with temporal food consumption. CR is known to retard aging and extend life span of animals via yet-unknown pathways. We hypothesize that resetting the biological clock could be one possible mechanism by which CR extends life span. Because it is experimentally difficult to uncouple calorie reduction from temporal food consumption, we took advantage of the murine urokinase-like plasminogen activator ({alpha}MUPA) transgenic mice overexpressing a serine protease implicated in brain development and plasticity; they exhibit spontaneously reduced eating and increased life span. Quantitative real-time PCR analysis revealed that {alpha}MUPA mice exhibit robust expression of the clock genes mPer1, mPer2, mClock, and mCry1 but not mBmal1 in the liver. We also found changes in the circadian amplitude and/or phase of clock-controlled output systems, such as feeding behavior, body temperature, and enteric cryptdin expression. A change in the light-dark regimen led to modified clock gene expression and abrogated circadian patterns of food intake in wild-type (WT) and {alpha}MUPA mice. Consequently, food consumption of WT mice increased, whereas that of {alpha}MUPA mice remained the same, indicating that reduced food intake occurs upstream and independently of the biological clock. Thus we surmise that CR could lead to pronounced and synchronized biological rhythms. Because the biological clock controls mitochondrial, hormonal, and physiological parameters, system synchronicity could lead to extended life span.

murine urokinase-like plasminogen activator; aging; biological clock; food; FVB/N; defensins; caloric restriction

THE MASTER CLOCK located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus in the brain regulates circadian rhythms in mammals. Similar clock oscillators have been found in peripheral tissues, such as the liver, intestine, retina, and peripheral blood mononuclear cells (3, 23, 39). A critical feature of circadian timing is the ability of the clockwork to be reset by light, with the retinohypothalamic tract being the principal pathway through which entraining information reaches the SCN (26). Environmental light, perceived by the retina, entrains the SCN clock to the 24-h day. Synchronization among the SCN neurons leads to coordinated circadian outputs from the nuclei, ultimately regulating cellular and physiological systems, including energy metabolism in the liver, sleep-wake cycles, and rhythms in hormone secretion (16, 24, 36, 55). A number of genes constitute the evolutionarily highly conserved biological clock. Clock, the first clock gene identified in mammals (53), encodes a transcription factor, CLOCK, that dimerizes with BMAL1 to affect transcriptional activation. Thus CLOCK and BMAL1 constitute the positive limb of the clock (39). Per1 and Per2 together with Cry1 and Cry2 are induced by CLOCK and BMAL1, but once the proteins are produced, they inhibit transcription and serve as the negative limb of the clock (8, 39). Pers and Bmal1 have robust oscillation, although in opposite phases, correlating with their opposing functions (15, 56).

The prominent influence of the circadian clock on human physiology and pathophysiology occurs primarily at the transcriptional level and is demonstrated by the temporal variations in hormone levels, pharmacokinetics, and some aspects of disease (39). Disruptions in the circadian rhythms have led to cancer proneness and reduced life expectancy (7, 11, 19, 20, 22, 38). Moreover, resetting of circadian rhythms has led to increased longevity (19).

Caloric restriction (CR) is an intervention that extends the life span of diverse species, such as nematodes, fruit flies, and rodents. CR in mice and rats extends youth and prevents or delays the onset of major age-related diseases, such as cancers, diabetes, kidney disease, cataracts, etc. (27, 54). Recent findings indicate that calorically restricted monkeys exhibit better health and lower disease risk compared with controls (42, 43). The reduction of energy intake is considered to be the critical beneficial factor in the CR regimen. However, the mechanisms by which CR modulates aging and longevity are virtually unknown (27). The most prevalent is the free radical/oxidative stress theory of aging, which attributes the aging-linked deterioration to the continuous accumulation of oxidative damage generated by reactive oxygen species produced in the mitochondria (14). Accordingly, CR was suggested to increase the resistance to oxidative stress and reduce oxidative damage (54). More recently, CR was suggested to ameliorate the consequences of oxidative damage, for example, by enhancing apoptosis (37, 46, 50, 51).

When food is available only for a limited time each day, with no CR, mice and rats adjust to the feeding period within a few days and learn to eat their daily food intake as before the restriction (12, 18). In addition, the animals increase their locomotor activity 2–4 h before the onset of food availability. Such anticipatory behavior also occurs in other mammals and birds and is often paralleled by increases in body temperature, adrenal secretion of corticosterone, gastrointestinal motility, and activity of digestive enzymes (5, 45). Moreover, this protocol, known as restricted feeding (RF), resets the expression of clock genes in peripheral tissues but not in the SCN. RF occurs independently of the light-dark cycle, in constant light, and in animals with lesioned biological clock in the brain (34, 35, 47). Calorically restricted animals, fed once a day or every other day, resemble RF-treated animals, as they usually consume all or most of their food within a short period of time. Interestingly, however, CR protocols entrain the clock in the SCN (4, 29, 40), suggesting that calorie reduction per se could be involved in master clock modulation in addition to the temporal food intake. Furthermore, calorically restricted animals show a rise in body temperature while anticipating food (6), a response likely to be controlled by the biological clock. To the best of our knowledge, the effect on the biological clock has not been studied as a possible factor underlying the beneficial influence of CR.

Experimentally, it is difficult to eliminate the effect of RF, i.e., temporal food consumption, in calorically restricted animals. Therefore, to investigate the contribution of the biological clock in the context of reduced eating, we used {alpha}MUPA mice (30). These transgenic mice overexpress in the brain the urokinase-type plasminogen activator (uPA), a serine protease implicated in brain development and plasticity (1, 31, 49). {alpha}MUPA mice spontaneously eat less when fed ad libitum and live longer compared with their wild-type (WT) control mice (31). Two transgenic lines that eat less are available, {alpha}MUPA and {alpha}MUPA/15, thus implicating the transgenic enzyme causing reduced eating (33). {alpha}MUPA mice exhibit similarities to calorically restricted mice, such as reduced body weight and reduced level of serum IGF-I or glucose, enhanced capacity to conduct apoptosis, and reduced incidence of spontaneous or carcinogen-induced tumors or preneoplastic lesions in several tissues (50, 51). Therefore, {alpha}MUPA mice can serve as a model for CR and the increased life span that occurs in the absence of temporal food consumption. Here, we show that the expression of biological clock genes in {alpha}MUPA is more pronounced than in WT controls and that, most probably, as a result, so is the amplitude and/or phase of other clock output systems.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, treatments, and tissue. Female FVB/N, {alpha}MUPA, and {alpha}MUPA/15 mice (30–32) were obtained from the Weizmann Institute of Science (Rehovot, Israel) at 4–6 mo of age. The mice were housed in a temperature- and humidity-controlled facility (23–24°C, 60% humidity). Mice were entrained to a 12:12-h light-dark cycle (12:12-h LD) for 2 wk with food available ad libitum. Body temperature, food intake, and tissue collection were performed after the 2-wk acclimation. Rectal body temperature was measured using a thermometer (Huger). For normal clock and cryptdin gene expression, liver and jejunum tissues of mice fed ad libitum were collected every 3 h around the circadian cycle. Tissues were removed, as is routinely performed, under dim red light on the first day of total darkness (DD) to avoid masking effects by light. Every 3 h, mice, the livers and jejunums of which were about to be removed, were anesthetized with an intraperitoneal injection of ketamine-xylazine (100/7.5 mg/kg). Animals were humanely killed at the end of the experiment. For the RF experiments, after 2 wk of ad libitum feeding, mice were given food between ZT21 and ZT24 for 3 wk (ZT0 is the time of lights-on). The amount of food eaten was measured, and after 3 wk, the mice were killed and their livers removed around the circadian cycle under dim red light on the first day of total darkness (DD). For biological clock disruption, the light-dark cycle was changed every 8 h, i.e., there were 3 light-dark cycles in 24 h for 3 wk (LDLDLD). Body temperature, food intake, and tissue collection were performed after the 3-wk acclimation in the new light regimen. All tissues removed were analyzed by quantitative real-time PCR. The experiments were conducted in full compliance with the strict guidelines of The Hebrew University policy on animal care and use. A license to conduct this animal research was granted by our institutional offices.

RNA extraction and quantitative real-time PCR. For clock and cryptdin gene expression analyses, RNA was extracted from liver and small intestine, respectively, using TRI Reagent (Sigma). Total RNA was DNase I treated using RQ1 DNase (Promega) for 2 h at 37°C, as was previously described (10). Two micrograms of DNase I-treated RNA were reverse transcribed using MMuLV RT (Promega) and random hexamers; 1/20 of the reaction was then subjected to quantitative real-time PCR using the Sybr Green Master kit (Applied Biosystems) and the ABI Prism 7300 Sequence Detection system. Primers for Cryptdin 1, mPer1, mPer2, mCry1, mClock, and mBmal1 (F, forward; R, reverse; mCrypt1-F 5'-ttggagacccagaaggcactt-3', mCrypt1-R 5'-ccagatctctcaacgattcctctt-3'; mPer1-F 5'-ccgaatacacacttcgaaaccag-3', mPer1-R 5'-tcccgtttgcaacgcag-3'; mPer2-F 5'-cgggctatgaagcgcctag-3', mPer2-R 5'-ggttgttgtgaagatcctcttctca-3'; mCry1-F 5'-agccagctgatgtatttccca-3', mCry1-R 5'-agtttagtgatgttccattccttgaa-3'; mClock-F 5'-cctagaaaatctggcaaaatgtca-3', mClock-R 5'-ccttttccatattgcattaagtgct-3'; mBmal1-F 5'-caagaatgcaagggaggcc-3', mBmal1-R 5'-ttgtcccgacgcctctttt-3') were tested alongside the normalizing gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (mGapdh-F 5'-caagaggtggacacagtggaga-3', mGapdh-R 5'-cggccactatattcttcaaggc-3').


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Clock gene expression in {alpha}MUPA vs. WT mice. To study the biological clock of {alpha}MUPA mice, we tested the phase of the clock genes mPer1, mPer2, mCry1, mClock, and mBmal1 at the RNA level. Mice were entrained to a light-dark cycle for 2 wk, and their livers were removed on the first day of DD around the circadian cycle. Real-time PCR analyses revealed that all clock genes oscillated, as was previously reported (23). However, mPer1, mPer2, mCry1, and mClock exhibited higher levels in the {alpha}MUPA lines than in WT (Fig. 1). It is noteworthy that mPer1 and mPer2, but not mCry1 and mClock, displayed a more pronounced difference between peak and trough (20- to 25-fold) in the {alpha}MUPA lines compared with WT (10-fold) (Fig. 1). Notably, the mBmal1 expression pattern was similar in {alpha}MUPA and WT mice (Fig. 1).


Figure 1
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  		Fig. 1. Expression levels of mPer1, mPer2, mCry1, mClock, and mBmal1 in the liver of {alpha}MUPA, {alpha}MUPA/15, and wild-type (WT) mice. Total RNA extracted from liver tissue collected every 3 h around the circadian cycle (values are means ± SE; n = 3 for each time point and each mouse group) was reverse transcribed and analyzed by quantitative real-time PCR. Clock gene levels were normalized using Gapdh as the reference gene. The gray and black bars designate the subjective day and night, respectively. All clock genes tested oscillated during the circadian cycle (1-way variance, P < 0.0001). All clock genes tested except mBmal1 exhibited more robust levels in {alpha}MUPA and {alpha}MUPA/15 mice compared with WT.

 
Circadian patterns of food intake and body temperature of {alpha}MUPA vs. WT mice. In light of the differences in clock gene amplitude, we analyzed two clock-controlled output systems, food intake and body temperature, throughout the circadian cycle. To determine the eating pattern, mice were entrained for 2 wk in 12:12 LD, and ad libitum food intake was measured. As expected, the total food consumption of {alpha}MUPA and {alpha}MUPA/15 was relatively low, 56% (2.05 g·mouse–1·day–1) and 79% (2.89 g·mouse–1·day–1), respectively, compared with the WT control (3.66 g·mouse–1·day–1). However, the eating pattern of {alpha}MUPA and {alpha}MUPA/15 was more pronounced than that of WT (Fig. 2A). The pronounced feeding phase started toward the end of the subjective day, with a trough toward the second half of the subjective night, in accordance with previous results (33). In total, {alpha}MUPA mice ate less than WT mice during the dark phase (2.37 g/mouse for WT, 1.46 g/mouse for {alpha}MUPA, and 1.76 g/mouse for {alpha}MUPA/15). In addition, both {alpha}MUPA and {alpha}MUPA/15 had a small peak of eating in ZT3. In accordance with their food consumption, the body weight values of the mice were on average 28.8, 22.5, and 24.1 g for WT, {alpha}MUPA, and {alpha}MUPA/15, respectively. In light of the pronounced feeding phase, we measured body temperature throughout the circadian cycle. As expected, rectal body temperature correlated with the feeding behavior in phase and amplitude in {alpha}MUPA lines with a "major drop" at the second half of the subjective night. Notably, there was an ~1.5°C difference between peak and trough in the transgenic mice, with only a 0.5°C difference in WT mice (Fig. 2B). Thus body temperature and feeding behavior, two outputs of the biological clock, exhibit pronounced and shifted phases in {alpha}MUPA compared with WT mice. The body temperature of {alpha}MUPA mice was not lower than that of WT mice, as was previously reported (33), most likely because of differences in ambient conditions in the animal facility.


Figure 2
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  		Fig. 2. Food consumption and body temperature in {alpha}MUPA, {alpha}MUPA/15, and WT mice. Food consumption (A) and body temperature (B) were measured every 3 h around the circadian cycle (n = 10 for each mouse group; values are means ± SE). Food consumption and body temperature exhibited a peak at the end of the subjective day to the first half of the subjective night in {alpha}MUPA and {alpha}MUPA/15 mice (1-way variance, P < 0.0001). WT mice exhibited a shallow peak throughout the circadian cycle with statistical significance (1-way variance, P < 0.0001).

 
Circadian patterns of cryptdin gene expression in {alpha}MUPA vs. WT mice. To study a clock output system at the molecular level, we examined cryptdin oscillatory expression in {alpha}MUPA and WT mice. We have recently shown that cryptdins, antibacterial polypeptides secreted from Paneth cells of the small intestine, exhibit oscillatory expression throughout the circadian cycle, peaking toward the end of the subjective night (9). To study whether cryptdin expression was also shifted, mice were entrained for 2 wk in 12:12 LD and were euthanized on the first day of DD with their small intestine removed. Real-time PCR analyses of two parts of the small intestine, jejunum (Fig. 3) and ileum (data not shown), revealed that Cryptdin 1 (Fig. 3) and Cryptdin 4 (data not shown) indeed oscillate. However, cryptdin phase was different in the various mice groups ({alpha}MUPA, {alpha}MUPA/15, and WT) (Fig. 3). Thus these results showed that not only physiological clock outputs, such as feeding behavior (Fig. 2A) and body temperature (Fig. 2B), but also molecular outputs are affected, most probably, as a result of a more pronounced clock gene expression (Fig. 1) of {alpha}MUPA transgenic lines.


Figure 3
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  		Fig. 3. Cryptdin 1 expression levels in the jejunum of {alpha}MUPA, {alpha}MUPA/15, and WT mice. Total RNA extracted from jejunum tissue collected every 3 h around the circadian cycle (values are means ± SE; n = 3 for each time point and each mouse group) was reverse transcribed and analyzed by quantitative real-time PCR. Cryptdin 1 levels were normalized using Gapdh as the reference gene. The gray and black bars designate the subjective day and night, respectively. Cryptdin 1 oscillated during the circadian cycle (1-way variance, P < 0.0001) and exhibited a slight shift in the {alpha}MUPA lines.

 
RF of {alpha}MUPA vs. WT mice. Because the biological clock of {alpha}MUPA mice is more pronounced, we wanted to examine whether these mice eat less at night because of a major drop in clock function toward the end of the subjective night (Fig. 2). Therefore, we tested whether a shift in their biological clock, expected to result from food availability at the nadir of their eating time, would require them to eat more with no "switching off" of their biological clock. We used the RF protocol, in which food is available ad libitum but for a limited time during the circadian cycle. The food was introduced to {alpha}MUPA and WT mice at ZT21 (3 h before lights-on) for 3 h in the course of 3 wk. As expected, the mice exhibited food anticipatory behavior, and their clock gene expression shifted after a few days (data not shown), as has been extensively established (13, 48). Surprisingly, after approximately a week, both {alpha}MUPA and WT mice ate 93% (2.45 g·mouse–1·day–1 vs. 2.54 g/day) and 96% (3.77 g·mouse–1·day–1 vs. 4.05 g/day), respectively, of the total daily amount as before the start of the RF (Fig. 4). This behavior lasted for at least 2 wk (Fig. 4). Thus, although the biological clock of {alpha}MUPA mice was shifted in this experiment, as indicated by clock gene expression and eating behavior, they did not eat more, suggesting that the transgenic mice, like the WT mice, have an intrinsic capacity to "remember" their daily amount of food intake.


Figure 4
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  		Fig. 4. Food consumption during restricted feeding (RF) of {alpha}MUPA and WT mice. After 7 days of ad libitum food availability, food was given ad libitum for only 3 h between ZT21 and ZT24. At the end of the 3 h, the amount of food consumed was measured (n = 10 for each mouse group).

 
Clock disruption and eating behavior of {alpha}MUPA vs. WT mice. To further study the relationship between the biological clock and eating behavior of the mice, we examined food intake under conditions of disrupted biological rhythms. After 2 wk in 12:12 LD, the lighting cycle was changed every 8 h, i.e., there were three light-dark cycles in 24 h for 3 wk (LDLDLD). This light regimen is expected to disrupt the biological clock in the brain and consequently in peripheral clocks. Indeed, clock gene analysis by quantitative real-time PCR revealed that mPer1, mPer2, mCry1, and mClock exhibited no oscillations throughout the circadian cycle (data not shown), verifying clock disruption in both WT and {alpha}MUPA mice. Likewise, clock disruption abrogated the circadian pattern of food consumption in both {alpha}MUPA and WT mice (data not shown). Mouse daily food intake and body weight measurements revealed that WT mice gained weight (120% of their initial weight), and their daily food intake increased (140% of the initial intake) throughout the 3 wk (Fig. 5). Surprisingly, {alpha}MUPA mice maintained their low food intake as well as their reduced body weight (Fig. 5). Thus disrupted biological rhythms lead to higher food consumption in WT but not in {alpha}MUPA mice.


Figure 5
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  		Fig. 5. Food consumption and body weight of {alpha}MUPA and WT mice during clock disruption. After 2 wk in 12:12-h light-dark cycle (12:12-h LD), the lighting cycle was changed every 8 h, i.e., there were 3 light-dark cycles in 24 h for 3 wk (LDLDLD). During the 3 wk, daily food consumption (A) alongside body weight (B) of WT and {alpha}MUPA mice was measured (n = 10 for each mouse group). Arrow indicates the start of the LDLDLD regimen.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data show that {alpha}MUPA mice exhibit higher amplitude in the circadian expression of the clock genes in the liver compared with WT control mice (Fig. 1). This change coincides with more pronounced rhythms of food intake and body temperature (Fig. 2) and with shifts in cryptdin gene expression (Fig. 3). The most prominent difference between WT and {alpha}MUPA mice is food intake at the end of the dark phase. WT mice show low amplitude encompassing the entire dark period, whereas the two transgenic lines show sharp amplitude in the first half of the dark phase with very little eating during the second half (Fig. 2). Because circadian patterns of food intake, body temperature, and cryptdin gene expression constitute clock-controlled output systems, it is conceivable that their alteration in the transgenic mice stems from a more robust clock gene expression in the periphery (Fig. 1) and possibly also the central biological clock in the SCN.

Unlike more robust rhythmicity in the aforementioned output systems, {alpha}MUPA did not differ from WT mice in their capacity to respond to RF (Fig. 4), a regimen known to entrain peripheral clocks (13, 48). The fact that {alpha}MUPA mice ate the food within the late subjective night, during which they normally show minimal eating, implies that their peripheral biological clock can be entrained by food. {alpha}MUPA and WT mice learned within approximately a week to eat in 3 h the daily amount of food consumed before the start of RF regimen (Fig. 4). Even though their biological clock was shifted to the time of food availability, {alpha}MUPA "remembered" the lower amount of food and did not exceed the daily amount. These findings are in concert with those in other systems, in which RF did not lead to overall reduction in food consumption and the animals learned to consume faster their normal daily intake in a few days (12, 18). The finding that both WT and {alpha}MUPA mice responded similarly to the RF treatment, while maintaining their original amount of daily eating, could indicate that the mechanisms controlling the temporal and quantitative aspects of food intake are separate. Similar to the results achieved with the RF regimen, the amount of food intake was not changed in {alpha}MUPA mice even when the biological clock was disrupted (Fig. 5). Thus, whereas temporal food intake is controlled by the biological clock, the amount of food could be dictated by an upstream mechanism that can entrain the central biological clock, as has been previously shown for calorically restricted animals (4, 29, 40).

The changes related to the clock and food intake were detected in two {alpha}MUPA transgenic lines, thus pointing to uPA, the product of transgenic expression, as the primary causative factor. uPA is a secreted proteolytic enzyme that can play a major role in extracellular proteolysis and tissue remodeling and has been implicated in brain development (49), seizure-related plasticity (31), and cocaine-induced behavioral changes (1). Therefore, it can be assumed that uPA overexpression exerts irreversible structural changes in the developing or adult brain, leading to the aforementioned changes. For example, the two transgenic lines overexpress uPA specifically in the trigeminal nucleus of the brain stem, a region devoid of uPA expression in the WT brain (33). Thus, although this region has not been associated so far with the biological clock or food intake, it is plausible that the transgenic expression in the trigeminal nucleus is responsible for the changes detected in {alpha}MUPA. Recent results have shown increased levels of serotonin, a neurotransmitter affecting satiety, in the {alpha}MUPA brain, suggesting that serotonin could be largely responsible for the reduced food intake of {alpha}MUPA (Y. Avraham and R. Miskin, unpublished observation). In turn, the reduced food intake could affect the biological clock, as indicated by the SCN entrainment of CR mice (4, 29, 40).

The pronounced circadian rhythms shown here for {alpha}MUPA and previously reported for calorically restricted animals (4, 29) pose the biological clock as a possible major factor determining temporal feeding behavior, body temperature, mitochondrial activity, and many other bodily systems that can contribute to life span (Fig. 6). For example, as the clock controls the temporal and pronounced expression of IGF-I-binding protein (IGFBP-1) (17, 41), it could contribute to the reduction in serum levels of free insulin or IGF-I. Low levels of insulin or IGF-I in the blood are thought to be beneficial in the aging process (17, 25, 21, 41, 50). The importance of the biological clock in this context is further corroborated by the recent findings that homozygous Clock mutant mice exhibit attenuated circadian feeding rhythms, hyperphagia, obesity, and a high incidence of metabolic syndrome (52). It is noteworthy that this behavior is in contrast with that of {alpha}MUPA mice, which display a pronounced eating rhythm concomitantly with reduced food intake and body weight and lack of obesity (33). In addition, it has been shown that pronounced circadian rhythms extend life span in the golden hamster (19). In turn, calorie intake, energy metabolism, RF, and CR also feed back to the biological clock (4, 13, 29, 40, 48) and help sustain the rhythms. For example, the redox state determined by the mitochondria affects the dimerization of two clock proteins, CLOCK and BMAL1 (44). Also, in monkeys, CR attenuates age-associated decline in melatonin levels (28), a hormone whose levels are controlled by the biological clock and, in turn, resets the biological clock (20). Thus temporal and pronounced expression of the biological clock, which leads to temporal and pronounced expression of output systems, appears to be more advantageous than continuous low-amplitude system activity.


Figure 6
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  		Fig. 6. Relationship between the biological clock, eating behavior, and longevity. Caloric restriction (CR) can entrain peripheral clocks via temporal feeding and possibly resets the central clock via calorie reduction. In turn, the reset biological clock leads to robust and temporal expression of genes involved in mitochondrial, hormonal, and metabolic functions, thereby affecting cellular and physiological processes. This robust and temporal expression eventually retards the aging process and extends life span.

 
In conclusion, the {alpha}MUPA lines showed a more robust biological clock than that of WT, as indicated by the expression of clock genes, along with more pronounced circadian patterns of food intake and body temperature. Given the {alpha}MUPA model and the reported capacity of CR to entrain the circadian clock, it appears that CR could influence longevity by resetting the clock, because of the reduced calories and possibly also the temporal feeding. Such clock adjustment can influence a plethora of systems among which are mitochondrial and endocrine activities. As a result, cellular and physiological systems perform in a more synchronized and robust manner. These changes can mitigate the level of molecular damage and ensure a better tissue homeostasis, which may attenuate the process of aging and increase longevity (Fig. 6). Indeed, several mitochondrial alterations previously described in {alpha}MUPA collectively suggest that superoxide dismutase-2, an inducible mitochondrial antioxidant enzyme, can modulate the balance between apoptosis vs. inflammation and carcinogenesis (50, 51). The interrelation between the biological clock and CR raises the question as to what extent each factor contributes to longevity. This issue can be further assessed by measuring the life span of clock mutant animals and mice treated for RF, or measuring the biological clock of mouse models showing extended life span (2), such as the Ames dwarf and mice deficient in IGF-I-related receptors.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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We thank Nutricia Research Foundation (grant no. 2006-02) for support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Froy, Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew Univ. of Jerusalem, PO Box 12, Rehovot 76100, Israel (e-mail: froy{at}agri.huji.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 

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